The present invention relates to the use of a vector which expresses DNA polymerase xcex2 as a DNA medicinal product in the context of the molecular treatment of cancer and viral diseases such as AIDS.
Essentially two types of genetic treatment exist for these diseases, namely immunotherapy and the introduction of xe2x80x9csuicidexe2x80x9d genes by viral vectors into the target cells.
Genetic immunotherapy is the use of tumor-infiltrating lymphocytes (TILs), which are highly tumoricidal. When they are transformed, they convey interleukin (IL2, IL4, IL6, xcex3-interferon) genes to the tumor and these activate the immune response locally, allowing both a limitation of the side effects on the body and an amplification of their antitumor effect.
The approach directed toward transferring a xe2x80x9cmedicinal DNAxe2x80x9d or therapeutic gene into a tumor cell has already been proposed (Culver et al., 1994) in the context of the use of genes such as the HSV tk gene of thymidine kinase from the human herpesvirus HSV-1 (Moolten et al., 1990; Culver et al., 1992) or VZV tk if it is from the chickenpox virus (Huber et al., 1991), the bacterial genes gpt coding for xanthine/guanine phosphoribosyl-transferase (Mroz et al., 1993) or codA coding for cytosine deaminase (Mullen et al., 1992). The products of these xe2x80x9csuicidexe2x80x9d genes convert initially nontoxic agents, such as ganciclovir (GCV) in the case of HSV-TK, 6-methoxypurine (ara-M) with VZV-TK, 5-fluorocytosine (5-FC) with CodA and 6-thioxanthine (6-TX) with GPT, into products which are highly toxic to the cell.
Viral thymidine kinase (TK) genes have been used in particular to destroy several types of cancer cells (Moolten et al., 1990; Huber et al., 1991; Vile et al., 1993), making these cells sensitive to purine or pyrimidine analogs such as acyclovir (ACV), ganciclovir (GCV) or bromovinyldeoxyuridine (BVDU). These nucleoside analogs are converted by the viral TKs into diphosphorylated forms which are then triphosphorylated with endogenous cell enzymes before incorporation into the tumoral DNA by DNA polymerases. The incorporation of these chain terminators (absence of 3xe2x80x2 OH) blocks the replication of the DNA and leads to cell death.
The present invention proposes to use a novel type of suicide gene whose function consists in facilitating the incorporation of a nucleotide analog into the DNA of the target cell after phosphorylation of the nucleoside prodrug optionally by a xe2x80x9cstandardxe2x80x9d suicide gene.
The incorporation of nucleotides into DNA is naturally carried out in eukaryotic cells by DNA polymerases. Among the mammalian DNA polymerases, DNA polymerase xcex2 has a number of specific features.
DNA polymerase xcex2 is a polypeptide of 39 kD and is an enzyme which is highly conserved in higher eukaryotes (Kornberg et al., 1992). Its primary function is believed to be the repair of damaged DNA (Sobol et al., 1996), but it also has a role in the replication of native DNA (Jenkins et al., 1992; Sweasy et al., 1992). DNA polymerase xcex2 is expressed at a constant level during the cell cycle (Zmudzka et al., 1988) and exposure of the cell to xenobiotic agents such as radiations induces its expression (Srivastava et al., 1995; Fornace et al., 1989). It differs from the other polymerases in its small size and its unfaithful nature during DNA replication, this infidelity being linked to the absence of associated corrective exonuclease activities (Kunkel et al., 1986).
in vitro, it has been shown that DNA polymerase xcex2 incorporates ddCMP (triphosphorylated dideoxycytidine), an inhibitor of DNA synthesis, with an efficacy which is comparable to that observed for the incorporation of the natural antagonist dCMP or deoxycytidine monophosphate (Copeland et al., 1992). Very similar results have been published in relation to AZT (Copeland et al., 1992; Parker et al., 1991). in vivo, AZT-MP (azidothymidine monophosphate) is in fact incorporated into cellular DNA (Sommadossi et al., 1989) and it has been suggested that DNA polymerase xcex2 plays a role in this process (Parker et al., 1991).
Thus, a subject of the present invention is, in particular, the use of a vector which expresses DNA polymerase xcex2 or an analog of DNA polymerase xcex2 in order to incorporate nucleotide analogs with antiviral or antitumor activity into a cell""s DNA, for the manufacture of a medicinal product intended for the treatment of cancer or a viral disease such as AIDS.
The expression xe2x80x9cDNA polymerase xcex2 analogxe2x80x9d means any nucleotide sequence which has at least 80% homology with the nucleotide sequence of mammalian DNA polymerase xcex2 and which fulfils the same functions.
The present invention thus consists in inducing an intracellular overproduction of an enzyme which is normally present in low amount in the cell, namely DNA polymerase xcex2, in order to amplify its unfaithful and mutagenic nature and thus force the incorporation of nucleotide analogs into the DNA.
A subject of the present invention is also an expression vector comprising a gene coding for DNA polymerase xcex2 or an analog of DNA polymerase xcex2. The vector is intended to express DNA polymerase xcex2 in tumor cells or cells infected with a virus such as the AIDS virus. It is thus advantageous to place said gene under the control of an expression system which is effective in the target cells.
According to one advantageous embodiment of the present invention, the expression vector in accordance with the present invention comprises a target sequence and/or expression sequence which is specific for tissues in which it is desired to express the DNA polymerase xcex2 (or an analog), such as tumors or tissues infected with viruses; by way of example, mention may be made of hematopoietic strain cells for the eradication of CD4+ lymphocytes infected with the HIV virus.
The expression vector according to the present invention can be any vector commonly used in gene therapy, and particularly a viral vector derived from a virus chosen from adenoviruses, adeno-associated viruses, retroviruses (including HIV), herpesviruses, poxviruses, parvoviruses, plasmoviruses, Semliki Forest viruses and Sindbis viruses.
As indicated above, DNA polymerase xcex2 allows the incorporation of nucleotide analogs into DNA. Now, certain nucleoside analogs are known to have antiviral activity when they are phosphorylated, i.e. in the form of nucleotides. This is the case in particular for AZT and ddC. These nucleoside analogs are normally atoxic to cells. However, after phosphorylation and in the presence of DNA polymerase xcex2 (or analog), they are incorporated into DNA and block its replication. The phosphorylation in question can be carried out in particular by thymidine kinase and/or thymidilate kinase.
Thus, according to one particularly advantageous embodiment, the use of a vector which expresses DNA polymerase xcex2 (or analog) in accordance with the present invention can be potentiated by the expression, in these same target cells or tissues, of the thymidine kinase and/or thymidilate kinase gene.
The cells in which the DNA polymerase xcex2 and the thymidine kinase and/or thymidilate kinase are expressed are thus made more sensitive to nucleoside analogs such as AZT or ddC, which, in situ, are phosphorylated before being incorporated into the DNA.
The thymidine kinase and/or thymidilate kinase gene can be inserted on the same vector as the one which expresses DNA polymerase xcex2 or on another vector. In the latter case, the target cell will undergo a co-transfection in order to allow the expression of each of the genes concerned. In any case, the gene coding for thymidine kinase and/or thymidilate kinase will be placed under the control of an expression system which is effective in the target cells to be reached.
The subject of the present, invention is also cells transformed with an expression vector in accordance with the invention comprising a gene coding for DNA polymerase xcex2 (or analog) and optionally also comprising a gene coding for thymidine kinase and/or thymidilate kinase, as well as to the cells transformed with an expression vector in accordance with the invention comprising the two abovementioned genes.
Moreover, it should be pointed out that the vectors in accordance with the present invention can be used as cloning vectors. The insertion of a polylinker into the gene coding for DNA polymerase xcex2 (or an analog) without modifying the reading frame, which is itself inserted into a vector such as pUT-pol xcex2 or pZHTk xcex2, allows a gene of interest to be cloned. The recombinant cells transfected with this type of vector can readily be selected since they have become resistant to nucleoside analogs such as AZT or ddC following inactivation of the DNA polymerase xcex2 (or analog) gene.
Lastly, a subject of the present invention is a product containing an expression vector according to the invention comprising a gene coding for DNA polymerase xcex2 (or analog) and a gene coding for thymidine kinase and/or thymidilate kinase or two expression vectors each comprising one of the genes in question and at least one antiviral agent, as a combination product for simultaneous or separate use or for use spread out over time. Preferably, the antiviral agent is AZT or ddC.
It should also be noted that the use of expression vectors and/or cells transformed with these expression vectors, in accordance with the present invention in the context of the treatment of cancers or viral diseases such as AIDS, can be combined with any other standard treatment such as chemotherapy or radiotherapy.